title: Electrical stimulation promotes maturation of cardiomyocytes derived from human embryonic stem cells.
abstract: While human embryonic stem cells (hESCs) can differentiate into functional cardiomyocytes, their immature phenotypes limit their therapeutic application for myocardial regeneration. We sought to determine whether electrical stimulation could enhance the differentiation and maturation of hESC-derived cardiomyocytes. Cardiac differentiation was induced in a HES3 hESC line via embryoid bodies formation treated with a p38 MAP kinase inhibitor. Detailed molecular and functional analysis were performed in those hESC-derived cardiomyocytes cultured for 4 days in the absence or presence of electrical field stimulation (6.6 V/cm, 1 Hz, and 2 ms pulses) using an eight-channel C-Pace stimulator (Ion-Optics Co., MA). Upon electrical stimulation, quantitative polymerase chain reaction demonstrated significant upregulation of cardiac-specific gene expression including HCN1, MLC2V, SCN5A, SERCA, Kv4.3, and GATA4; immunostaining and flow cytometry analysis revealed cellular elongation and an increased proportion of troponin-T positive cells (6.3 ± 1.2% vs. 15.8 ± 2.1%; n = 3, P < 0.01). Electrophysiological studies showed an increase in the proportion of ventricular-like hESC-derived cardiomyocytes (48 vs. 29%, P < 0.05) with lengthening of their action potential duration at 90% repolarization (387.7 ± 35.35; n = 11 vs. 291.8 ± 20.82; n = 10, P < 0.05) and 50% repolarization (313.9 ± 27.94; n = 11 vs. 234.0 ± 16.10; n = 10, P < 0.05) after electrical stimulation. Nonetheless, the membrane diastolic potentials and action potential upstrokes of different hESC-derived cardiomyocyte phenotypes, and the overall beating rate remained unchanged (all P > 0.05). Fluorescence confocal imaging revealed that electrical stimulation significantly increased both spontaneous and caffeine-induced calcium flux in the hESC-derived cardiomyocytes (approximately 1.6-fold for both cases; P < 0.01). In conclusion, electrical field stimulation increased the expression of cardiac-specific genes and the yield of differentiation, promoted ventricular-like phenotypes, and improved the calcium handling of hESC-derived cardiomyocytes.
authors.affiliation: Cardiology Division, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong, Hong Kong SAR, China.
authors.forename: Yau-Chi
authors.initials: YC
authors.lastname: Chan
authors.forename: Sherwin
authors.initials: S
authors.lastname: Ting
authors.forename: Yee-Ki
authors.initials: YK
authors.lastname: Lee
authors.forename: Kwong-Man
authors.initials: KM
authors.lastname: Ng
authors.forename: Jiao
authors.initials: J
authors.lastname: Zhang
authors.forename: Zi
authors.initials: Z
authors.lastname: Chen
authors.forename: Chung-Wah
authors.initials: CW
authors.lastname: Siu
authors.forename: Steve K W
authors.initials: SK
authors.lastname: Oh
authors.forename: Hung-Fat
authors.initials: HF
authors.lastname: Tse
mesh_terms: D020013:Calcium Signaling; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D004558:Electric Stimulation; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D008564:Membrane Potentials; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D010641:Phenotype; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D047428:Protein Kinase Inhibitors; D048051:p38 Mitogen-Activated Protein Kinases